5{62 -taurocholenic acids and 5{62 -taurocholadienic acids in compositions for reducing the concentration of cholesterol and liads in blood serum

ABSTRACT

5 Beta -Taurocholenic acids wherein the double bond may be either in the 22-position of the aliphatic chain or within the cholane ring; and 5 Beta -taurocholadienic acids wherein one double bond is in the 22-position of the chain and the second is within the cholane nucleus; and the salts, esters and amide derivatives thereof. The products are hypocholesterolemics and hypolipemics which have utility in the treatment of conditions associated with blood lipid deposition. The choice of a suitable method for preparing the final products depends largely upon the type of unsaturation in the final product; however, each route bears in common the terminal step of treating a 5 Beta -cholenic acid with an alkyl haloformate in the presence of a base followed by the reaction of the mixed anhydride intermediate thus formed with taurine in a basic medium to yield a 5 Beta -taurocholenic acid salt or 5 Beta taurocholadienic acid salt and, if desired, the salt thus obtained may then be converted to its free acid, ester or amide by conventional means.

United States Patent [72] Inventors Arthur A. htcbett Cranford; John Hannah, Mltawan, both of NJ. [21 Appl. No. 677,080 [22] Filed Oct. 23, 1967 [45] Patented Nov. 23, 1971 [73] Assignee Merck 8: Co., Inc.

Rahway, NJ.

[54] SB-TAUROCHOLENIC ACIDS AND 53- TAUROCHOLADIENIC ACIDS IN COMPOSITIONS FOR REDUCING THE CONCENTRATION OF CHOLESTEROL AND LIADS IN BLOOD SERUM [56] References Cited OTHER REFERENCES Fieser, Louis F. et al., Steroids, Reinhold Publishing Corp. 1959) p. 423

Levene, P. A. et al., Chemical Abstracts, Vol. 25, pp. 4278- 4279 1931 Bremer, 1., Biochemical J. Vol. 63, pp. 507- 513 (1956) Primary Examiner-Stanley .l. Friedman Atmmeys-Joseph W. Molasky, J. Jerome Behan and I. Louis Wolk ABSTRACT: SB-Taurocholenic acids wherein the double bond may be either in the 22-position of the aliphatic chain or within the cholane ring; and 5fi-taurocholadienic acids wherein one double bond is in the 22-position of the chain and the second is within the cholane nucleus; and the salts, esters and amide derivatives thereof. The products are hypocholesterolemics and hypolipemics which have utility in the treatment of conditions associated with blood lipid deposition.

The choice of a suitable method for preparing the final products depends largely upon the type of unsaturation in the final product; however, each route bears in common the terminal step of treating a SIS-cholenic acid with an alkyl haloformate in the presence of a base followed by the reaction of the mixed anhydride intermediate thus formed with taurine in a basic medium to yield a SB-taurocholenic acid salt or SB-taurocholadienic acid salt and, if desired, the salt thus obtained may then be converted to its free acid, ester or amide by conventional means.

SB-TAUROCI-IOLENIC ACIDS AND 53- TAUROCI-IOLADIENIC ACIDS IN COMPOSITIONS FOR REDUCING THE CONCENTRATION OF CHOLESTEROL AND LIADS IN BLOOD SERUM This invention relates to a new class of chemical compounds which can be described generally as SB-taurocholenic acids and SIB-taurocholadienic acids and to the nontoxic, pharmacologically acceptable salts, esters and amide derivatives thereof.

Also, it is an object of this invention to describe a novel method of preparation for the instant products, their salts, esters and amides.

Studies show that cholesterol and triglyceride play a major role in the formation of atherosclerotic plaques by accelerating the deposition of blood lipids on the arterial wall. It is the purpose of this invention to disclose a new class of chemical compounds which effectively reduce the concentration of cholesterol, triglyceride and other lipids in blood serum and, therefore, ameliorate conditions associated with blood lipid deposition.

The SB-taurocholenic acids and SIS-taurocholadienic acids of this invention are compounds having the following general formula:

wherei ni is hydroxyialkoxy, for example, lower alkoxy such as methoxy, ethoxy, etc., --OM wherein M is a cation derived from a metal of the first group of the Periodic System as, for example, the cation derived from an alkali metal hydroxide, carbonate, nitrate, etc. such as sodium hydroxide, potassium hydroxide, potassium carbonate, silver nitrate, etc., or an amino radical of the formula: NR'R wherein R and R are similar or dissimilar members selected from hydrogen or lower alkyl such as methyl, ethyl, etc., and when R is hydroxy, the nontoxic, pharrnacologically acceptable acid addition salts of the resulting acid as, for example, the salts obtained by treating the said acid with a primary, secondary or tertiary amine, for example, methylamine, dimethylamine, triethylamine, etc. or with hydrazine, N,N-dimethylhydrazine, etc.

The dotted line which appears in the three-position of the foregoing formula (I) is meant to indicate that the instant products may or may not contain an unsaturated double bond within the cholane ring and indicates further that the point of nuclear unsaturation is not limited to the three-position in the ring but is intended to include all other isomeric derivatives thereof as, for example, the A, A, etc, isomeric derivatives.

The several varieties of products which are embraced by this invention can be further illustrated by the following planar formulae. Thus, for example, the SIS-taurocholenic acids (la, infra) are products having the following general formula:

wherein R Trans-Aas defined above and wherein it is understood that the A double bond can be in any one of several positions in the cholane nucleus as, for example, in the A" and A" positions, etc.

Also, included within this invention are those isomeric derivatives of SB-taurocholenic acid (Ia, supra) wherein the double bond is located solely within the carbocyclic nucleus. Typical of these derivatives are the A, A, A", A'" and A isomers illustrated by the planar formula the formula (lc) illustrates a A -52 acid product it is to be understood that the point of nuclear unsaturation is not limited to the three-position but includes as well the A, A", A and A isomeric derivatives thereof:

SO R

H Io

wherein R is as defined above. The foregoing products (lc) also effect a significant reduction in the concentration of cholesterol in blood serum and, therefore, like their A isomers, are similarly useful in the treatment of conditions associated with blood lipid deposition.

Although all of the instant products (I) effectively reduce the concentration of cholesterol and triglyceride in blood serum it has been found that the alkali metal salts of i -sptaurocholenic acid (ld, infra) are especially suitable for this purpose. This subclass of compounds combines a high order of activity with little or no adverse side effects and, therefore, represents a preferred subgroup within the scope of this invention. The following structural formula illustrates this preferred embodiment:

whereinjl is a cation derive dfrom a metal of t h e iirst group of the Periodic System as, for example, sodium, potassium, etc.

The presence of the A ethylenic double bond in the instant products (I) gives rise to geometrical isomerism, i.e., the possibility of a cis-trans arrangement of functional groups. The desired spatial arrangement is most advantageously achieved 1 by employing as a reactant in the preparative method to be discussed infra either the cis or the trans starting material corresponding to the desired isomeric product; however, it will be appreciated by those skilled in the art that other methods may j also be employed as, for example, by a separation of a cis and I trans mixture of the product (I) by conventional separation techniques. Both the cis and trans varieties of the instant compounds (I) are useful in the treatment of hypercholesterolemia and it is to be understood that both isomeric derivatives as '15 well as the stereoisomen'c mixtures of those isomers are within the scope of this invention. The SB-taurocholenic acids and SB-taurocholadienic acids iof this invention are conveniently obtained by treating an ap- ;propriate Sfl-cholenic acid (H, infra) or SB-choladienic acid with an alkyl haloformate in the presence of a base, followed by the reaction of the mixed anhydride (e.g., Ill, infra) thus ?obtained with taurine in a basic medium as, for example, in an gaqueous solution of an alkali metal hydroxide, etc. or in the fi esence of a trialkylamine, etc.; the SB-taurocholenic acid salt (is, mm) or fifi-t a urocholadienic acid salt thus formed may then be isolated and purified as the product of the invention or, if desired, the said salt may be converted to its corresponding sulfonic acid (e.g., If, infra) by the application of 30 conventional techniques as, for example, by running a solution of the salt (If) in aqueous dimethylformamide or in aqueous methanol through a sulfonated ion-exchange column followed by evaporation of the solvent in vacuo from the resulting elu- 1 ate. The following equation illustrates the foregoing method of 5 preparation, however, it is to be understood that the A -5,8- cholenic acid depicted below as the starting material (ll) in the process is only illustrative of the several types of isomeric derivatives which may be similarly employed; when, for example, a A, A, A, A" or A isomer of A-5B-cholenic acid (ll) is employed as a starting material in the process and the vreaction is conducted in an otherwise analogous manner the corresponding A, A, A', A and A-5B-taurocholenic acid product is obtained and when the appropriate A A' or A"--5B-choladienic acid, etc. is substituted for the said A -5 fi-cholenic acid reactant (ll) the corresponding A A and A -5a'fl-taurocholadienic acid product, etc. is obtained:

I? C\ 503M W H Ie Ion Exchange l 9 (L1\ SOIH W wherein R is lower alkyl, for example, methyl, ethyl, etc. and M is as defined above.

Also, in lieu of taurine in the foregoing equation, a taurine amide may be substituted therefor and the process conducted in an otherwise similar manner to yield the corresponding 5;:- cholenic acid taurine amide (IV, infra) or SB-choladieaic acid taurine amide. However, when taurine amide is'aubstituted for the taurine starting material it should, of course, be understood that the ion-exchange step of the preceding equation is no longer required because the taurine amide reacts directly with the mixed anhydride intennediate (III), to yield the desired Sfi-cholenic acid taurine amide (IV). The following equation illustrates this method of preparation:

[I Base ClCOR 0 lloom H,NCH,-CH,S OgNHgl BISQ Yvk gv S O NH picted therein and conducting the reaction in an otherwise analogous manner.

The A -5B-cholenic acids (II) employed as starting materials in the foregoing methods of preparation are conveniently obtained by treating SB-cholanic acid (V, infra) with an appropriate halogenating agent and then with an alkanol to yield the corresponding alkyl 5B-23-halocholanate (VI, infra) and the ester intermediate thus obtained is then dehydrohalogenated to its corresponding alkyl A -SB- cholenate (Vll, infra) by treatment with a suitable reagent as, for example, by treatment with sodium bromide and calcium carbonate in dimethylformamide, followed by the hydrolysis of the said ester (Vll) to its corresponding acid by treating the former with an aqueous alcoholic solution of a base and then with an acid to yield the desired A -5fi-cholenie acid (H). The following equation, wherein the halogenating agent employed is bromine in a mixture of carbon tetrachloride and phosphorus tribromide and wherein the dehydrohalogenating agent is sodium bromide and calcium carbonate in dimethylformamide, illustrates the foregoing method of preparation; however, it is to be understood that other functionally equivalent halogenating and dehydrohalogenating agents may be substituted therefor and the process conducted in an otherwise similar manner to yield an identical A -5fi-cholenic acid (H):

Br; R OH C Cit/FBI;

I? COR l Br H VI

NaBr/CaChl DMF I? COR H VII Hydrolysis l wherein R is alkyl, for example, lower alkyl such as methyl,

ethyl, etc. and ROH is an alkanol, for example, a lower al-; kanol such as methanol, ethanol, etc. Also, in lieu of converting the alkyl 5B-23-halocholanate intermediate (Vl, supra) to its corresponding alkyl E SE-cholenate (Vll, supra) it is possible to hydrolyze the former (i.e., VI) in the conventional manner by treatment with an aqueous solution of a base to yield a 5p-23-halocholanic acid. The halocholanic acid compound thus obtained also exhibits hypocholesterolemic activity and, therefore, can also be used in the treatment of conditions associated with blood lipid deposition.

The foregoing is a convenient method for the preparation of the trans-A -5B-cholenic acid starting materials (ll) but, unfortunately, it is not suitable for the preparation of the corresponding cis isomers. The said cis-A -5fi-cholenic acids are obtained by treating 23-nor-24-hydroxy-5B-cholane (VllI, infra) with a suitable oxidizing agent as, for example, with a mixture of chromium trioxide and sulfuric acid and the 23-nor-24- oxo-SB-cholane (IX, infra) thus obtained is then halogenated 1 to the corresponding 23-nor-24-dihalo-5B-cholane (X, infra) and the latter subjected to dehydrohalogenation to yield 20aethynyl-SB-pregnane (XI, infra); the ethynyl compound (XI) thus obtained is then treated with methyl lithium in the presence of carbon dioxide and then with an aqueous solution of an acid such as hydrochloric acid to yield a 22-5B-cholynic acid (XII, infra) which is then subjected to catalytic hydrogenation as, for example, by treating the 22-5fi-cholynic acid (XII) with hydrogen in the presence of a lead-poisoned palladium on calcium carbonate catalyst, i.e., a Lindlar catalyst, to yield the desired cis-A -SB-cholenic acid (Ila). The following equation illustrates this method of preparation; however, the phosphorus pentachloride halogenating agent, sodamide dehydrohalogenating agent and hydrogenation catalyst depicted therein are only illustrative of the wide variety of reagents which may be employed and it will be apparent to those skilled in the art that other functionally equivalent reagents may be substituted therefor and the process conducted in an otherwise analogous manner to yield an identical cis-A -5B-cholenic acid compound:

CHIOH CHO Grog/H 804 VIII IX PClsl CHO], l x NINE] l 389 VCECH H XI LICHa/COgl HUI/H O CEGCO OH H XII Hgl Pb/Pd/QB(CO3)| OOH H IIa obtained by treating an appropriate nuclear hydroxy subsuch as acetic anhydride in pyridine andthe'res ulting nuclear alkanoyloxy substituted SB-cholanic acid (XIV, infra) is then treated with an appropriate halogenating agent and then with an alkanol to yield the corresponding alkyl alkanoyloxy 53-23 -halocholanate (XV, infra), which intennediate is then dehydrohalogenated to its corresponding alkyl alkanoyloxy trans-A -5/3-cholenate (XVI, infra) by treatment with a suitable reagent as, for example, by treatment with sodium bromide and calcium carbonate in dimethylformamide; the said alkyl alkanoyloxy Irans-A -SB-chQIenate (XVI) thus obtained is then hydrolyzed by treatment with an aqueous alcoholic solution of a base and then with an acid to yield the corresponding nuclear hydroxy substituted trans-A"-5fl-cholenic acid (XVII, infra) which is then esterified by treatment with a lower alkanol in a strong acid such as anhydrous hydrochloric acid to yield an alkyl nuclear hydroxy substituted trans-A SB-chOIenate intermediate (XVIII, infra); the cholenate intermediate (XVIII) thus obtained is then converted to its cor- ,former (i.e., XVIII) with paratoluenesulfonyl chloride in pyridine and the said sulfonate intennediate (i.e., XIX) is then converted to its corresponding A -trans-A-5fi-choladienic acid (XX, infra) by treatment with potassium tert.-butoxide in stituted SB-cholanie acid (XIII, infra) with an aeylating agent 5 a suitable solvent, such as dimethylsulfoxide, at raised temperatures. The following equation, wherein the cholanic acid starting material employed is 3g:hydroxy-5B-cholani c a c i d (XIII), illustrates the process; however, it will be appreciated by those skilled in the art that 7a-hydroxy-5B-cholanic acid and l2a-hydroxy-5B-cholanic acid, etc., may be substituted for the said 3a-hydroxy-5B-cholanic acid and the reaction conducted in an otherwise analogous manner to yield the corresponding A"-trans-A-5fi-choladienic acid, A -transA -5flcholadienic acid, etc.:

COOH C XIII Acetic Anhydrlde/Pyridlne llll COOH Br l CCll/PBI';

i L- llll NaBr/CaC DMF CH OHl I? COR 0 ens-60m;

KOHIHgOl HCll GIL-Q8 moi ridine l j GHQ-S otmw H Potassium tert.-butoxidel COOH H XX ivfiaeinwand R OH areas defined above.

There is no clear agreement about the actual role of cholesterol and triglyceride synthesis in the localization of atherosclerotic plaques but numerous studies support the concept that cholesterol and triglyceride play a major role in the pathogenesis of atherosclerosis because along with other lipids and fibrin they accumulate in the arterial intima and subintima to produce arterial corrosion.

Cholesterol and triglycerides are present to some extent in all ordinary diets and, also, they are synthesized by body organs from intermediates of metabolic origin; consequently, the development of a chemotherapeutic agent which will induce a significant reduction in the serum cholesterol and triglyceride level is considered desirable. To this end the 5B- taurocholenic acids and SB-taurocholadienic acids of this invention have been tested and found to exhibit good hypocholesterolemic and hypolipemic activity. The ability of the products to inhibit and reduce the concentration of cholesterol and other lipids in serum bespeaks their usefulness as pharrnacologically active compounds which have application in the treatment of conditions associated with cardiovascular disease.

The examples which follow illustrate the SB-taurocholenic and SB-taurocholadienic acids of this invention and the methods by which they are prepared. However, the examples are illustrative only and it will be apparent to those having ordinary skill in the art that all of the instant products may be prepared in an analogous manner by substituting the appropriate starting materials for those set forth in the examples.

EXAMPLE 1 Trans-A -5fl-Taurocholenic Acid Potassium Salt Step A: Methyl 5B-23-bromocholanate Bromine (3.6 ml.) is added slowly at room temperature to a mechanically stirred mixture of Sfi-cholanic acid (25.0 g.), carbon tetrachloride (100 ml.) and phosphorus tribromide (6.9 ml.) in a 250 ml. flask fitted with a reflux condenser topped by a drying tube containing anhydrous calcium sulfate. After 30 minutes, additional bromine 13.2 ml.) is added from a dropping funnel and the mixture is refluxed for 16 hours. The dark red solution is cooled to 0-5 C. and methanol (50 ml.) is added dropwise. Esterification is completed by refluxing the mixture for 1 hour. The mixture is then cooled to room temperature, diluted with carbon tetrachloride (100 ml.), decolorized with decolorizing carbon (2.5 g.), filtered and evaporated to dryness in vacuo. The residual gum is dissolved in a mixture of ethyl acetate (93 ml.) and methanol (93 ml.) and the flask scratched to induce crystallization. The mixture is then evaporated under reduced pressure in a rotary evaporator at 40 C. to about 100 ml. and more methanol (200 ml.) is added. The mixture is then cooled to 0- C. and filtered. The product is washed with cold methanol and dried in vacuo at 50 C. to yield methyl 5B-23bromocholanate (26.2 g., 85 percent) in the form of a yellow solid. Both diasteroisomeric forms of the product are present in substantially equal amounts. Analysis for Br: Calculated: 17.65 percent; Found: 17.69 percent. Step B: 5B-23-Bromocholanic Acid A solution of potassium hydroxide (0.381 g., 1 equivalent) in water (5 ml.) is added to a solution of methyl 513-23- bromocholanate (3.00 g.) in boiling ethanol (15 ml.). An cfily precipitate is immediately obtained but upon standing for approximately 1 minute the mixture becomes homogeneous. The solution is refluxed for 4 minutes, cooled to room temperature, acidified with aqueous concentrated hydrochloric acid and the precipate extracted with two 50 ml. portions of ether. The combined ehtereal solutions are then washed with water, dried over magnesium sulfate, filtered and evaporated in vacuo to yield crude 5/3-23-bromocholanic acid (2.87 g.). Recrystallization from hexane yields 1.44 g. of pure 53-23- bromocholanic acid, m.p. 172l 79 C.

A small sample of the product is purified for analysis by thili-layer'ch'ro'matography in chloroform over silica gel to yield highly pure B-23-bronsocholanic acid, m.p. 173-183' C ing da'k solution is cooled to room temperature, inorganic solution is cooled to room temperature, inorganic solution material is filtered ofl' and the solution diluted with water (2 liters) and extracted three times with 200 ml. portions of methylene chloride. The combined methylene chloride extracts are then washed with water (200 ml.), decolorized with decolorizing carbon (4 g.) at room temperature, filtered and evaporated to dryness in vacuo. The residual gummy methyl trans-A -5fl-cholenate thus obtained is dissolved in hot ethanol (300 ml.) and hydrolyzed by the addition of hot aqueous percent potassium hydroxide (400 ml.) with refluxing of the reaction mixture for 1 hour. The dark solution is then fcooled to room temperature and acidified to a pH of 2-3 with aqueous concentrated hydrochloric acid (100-120 ml.). EWater (400 ml.) is added to complete precipitation. The iproduct is filtered off, washed free of acid with water and ldried in vacuo at 80' C. to yield trans-A -5fl-cholenic acid {(15.0 g., 95 percent) in the form of a yellow solid, mp. Q l52'-l65' C. Purification by hot chromatography (45'-50' i C.) in benzene over silica gel, followed by crystallization from ether yields 60 percent recovery of trans-A-5/3-cholenic acid as a colorless solid, mp. l72-175 C.

Analysis for C,.H,,O,:

Calculated: C, 80.39; H, 10.68;

Found: C, 80.1 1; H, 10.76.

the methyl trans-A-5B-cholcnate intermediate described in the preceding preparative method has also been isolated, purified by chromatography over silica gel and crystallized from ethanol to yield pure compound having a melting point of 7 1'-74 C.

Analysis for G i-1, 0,:

Calculated: C, 80.60; H, 10.83;

Found: C, 80.45: H, 11.10.

Step D: mns-A dp-Taurocholenic Acid Potassium Salt Ethyl chloroformate (4.88 ml.) is added to a solution of rmns-A -5fi-cholenic acid (18.2 g.) in tetrahydrofuran (300 ml.) and triethylamine (7.88 ml.). After standing 2 hours at room temperature, precipitated triethylamine hydrochloride is filtered off and the solution evaporated to dryness in vacuo. Acetone (215 ml.) is added to the residual mixture of gummy mixed anhydride, followed by the addition of a solution of taurine (5.35 g.) in aqueous N-potassium hydroxide (43 ml.) and acetone (215 ml.). The mixture is stirred overnight at room temperature, cooled to 0-5 C. and. the crude product (16.9 g.) filtered oi! as a colorless solid. The trans-A-5B-taurocholenic acid potassium salt monohydrate thus obtained is then purified by twice recrystallizing from aqueous tetrahydrofuran followed by air-drying to yield 9.5 g. of, the 60.

said monohydrate in the form of a colorless solid. The product is then dried at 100 C. for 2 hours to yield anhydrous trans- A -5fl-taurocholenic acid potassium salt.

'" Analysis rSFc'j N863? Calculated: C, 61.98; H, 8.40; N, 2.78; S, 6.34; Found: C, 62.20; H, 8.43; N, 2.92; S, 6.56.

EXAMPLE 2 rrans-A -5fi-taurocholenic acid sodium salt monohydrate is mahmss'drv'wrrfivapiis of hot glacial acetic acid, filtered from insolubles and then precipitated at room temperature by the slow addition, with stirring, of 30 parts ofacetone. The product is filtered, washed with acetone 5 and dried in vacuo at 60 C. to yield 75 percent trans-A 53- taurocholenic acid sodium salt.

Analysis for C,,H ,NSO Na: Calculated: C, 64.03; H, 8.68; N, 4.72; Found: C, 64.28; H, 8.80; N, 4.53.

EXAMPLE 3 Trans-Ethyl A -5fi-Taurocholenate Step A: Trans-A -5fl-Taurocholenate Silver Salt A solution of silver nitrate (5.0 g.) in hot water 25 ml.) is added to a solution of trans-A -5fi-taurocholenic acid sodium salt (5.0 g.) in hot water (100 ml.) producing an immediate precipitate. The resulting precipitate is kept hot for minutes, cooled to room temperature and then filtered. The

silver salt is washed with water and dried in vacuo at 70' C. to

yield 4.80 g. of trans-A -5fi-taurocholenate silver salt.

Step B: Trans-Ethyl A-5B--Taurocholenate Silver A -5fi-taurocholenate (6.0 g.) is refluxed with an excess of ethyl iodide for 1 hour, cooled to 0 C. and silver iodide filtered ofi with methylene chloride washing and the comrocholenate, m.p. 1l21 14' C.

Analysis for C,,,H,-,NSO,: Calculated: C, 68.11;] 1, 9.59; N, 2.84; Found: C 68.20; H, 9.49; N, 2.87.

4 EXAMPLE 4 Trans-A-5fl-Cholenic Acid Taurine Amide Ethyl chloroformate (0.38 ml.) is added to a solution of rrans-A dfi-cholenic acid (1.44 g.) in tetrahydrofuran (20 ml.) and triethylamine (0.56 ml.) at0- C. and the anhydride intermediate thus obtained is maintained at the temperature for 1 hour. Taurine amide hydrochloride (0.642 g.) in water (4 ml.) containing triethylamine (0.56 ml.) is then added and the mixture is maintained at room temperature for 4 hours. The mixture is then evaporated to dryness in vacuo 5 product is then dissolved in tetrahydrofuran (9 ml.), filtered and petroleum ether (30 ml.) is added to precipitate the trans- A -5fi-cholenic acid taurine amide (1.2 g.), a colorless solid. The product is further purified by repeating the precipitation and drying the resulting solid in vacuo.

Analysis for C,.H N,SO,:

1 Calculated: C, 66.96; H, 9.68; N, 5.94;

Found: C, 67.20; H, 9.55; N, 6.03.

. EXAMPLE 5 A -5fl-Taurocholenic Acid Potanium Salt By substituting A -5fi-cholenic acid for the trans-A cholenic acid of example 1, Step .D, and following the procedure described therein the product A-5fi-taurocholenic acid potassium salt, m.p. 225-235 C. is obtained.

Analysis for C l'L NSOJCI Calculated: C, 61.98; H, 8.40;

Found: C, 61.87;]"1, 8.76.

EXAMPLE 6 A-5B-Taurocholenic Acid Potassium Salt By substituting A-5B-cholenic acid for the trans-A-5flcholenic acid of example 1, Step D, and following the procedure described therein the product A-5fi-taurocholenic acid potassium salt is obtained.

' EXAMPLE 7 p?e cipitate is vTashed with cold water and dried in vacuo to ld -t -A -5 h l d' 'd. A-5,6-Taurocholenic Acid Potassium Salt yle A mm B c o a lame ac! t G: A -T -A-5 -T h l d' A d P tass' By substituting A-5fi-cholenic acid for the trans-A 65- salst ep fans B auroc o a lemc c1 mm cholenic acid of example 1, Step D, and following the B a 22 t v. y substituting the A trans-A -5B-ch0ladiemc acid of Step procedure described therein the product A -SB-taurochoIemc F for the tranS An 5B cho1enic acid of example 1, Step D, and

I acld potassium Sams obtamed' following the procedure described therein the product A trans-A -5B-tauroch0ladienic acid potassium salt is obtained.

EXAMPLE 8 10 EXAMPLE ll A-5-Taurochol::i)c Acid Potassium Salt 22 A -Trans-A -5B-Taur0ch0ladienic Acid Potassium Salt By substituting A -5Bactd for the trans-A -5B-cholen1c= By substituting 7a hydroxy sfi cholanic acid for the 3% 5 acid exampl? Step fg the P 'hydroxy-SB-cholanic acid of example 10, Step A, and followdescnbcd therein the Product A 'sfitaurocholemc Mm 315 ing the procedure described in Steps A-G of the example the wiassium salt is wished i g product A'trans-A -5Btaurocholadienic acid potassium salt is F EXAMPLE9 f A-5B-Taurocholenic Acid Potassium Salt j i EXAMPLE 12 By substituting B- acid for the 6120; A"Trans-MSfi-Taurocholadienic Acid Potassium Salt 7 cholenic acid of example p D, and following the? By substituting l2a-hydroxy-5B-cholanic acid for the 301- P'P fiescribefi therein the Product An'sfl'taul'ocholenic .hydroxy-SB-cholanic acid of example 10, Step A, and follow- 5 and Potassium obtameding the procedure described in Steps A-G of that example the duvv" i 1 l product A -trans-A-5B-taurocholadienic acid potassium salt i EXAMPLE 10 1 I is obtained.

ATram-A-5B-Taurocholadienic Acid Potassium Salt Step A: 3oz-Acetoxy-5B-cholanic Acid EXAMPLE 13 A solution of 3a-hydroxy-5}3-cholanic acid lg.) in pyridine 1 i Cis-A -5j3-Tuarocholenic Acid Potassium Salt 2 (5ml.) and acetic anhydride (2ml.) is refluxed for 15 minutes f Step A: 23-Nor-24-oxo-5B-cholane then cooled to room temperature. Water (0.5 ml.) is added. 5 An aqueous 8N solution of chromium trioxide in sulfuric dropwise to hydrolyze the excess of acetic anhydride and any ia i -s equivalents) is added dropwise 0V6! a mixed anhydride- The reaction is moderated by cooling in; ;minute period to a vigorously stirred solution of 23-nor-24- cold water and after 10 minutes the warm solution is further y yfi- 8-- equivalents) in acetone diluted with water (4.5 ml.) to produce a crystalline 5 1 ml.) at 20' C. The mixture is then diluted with ether (100 ml.) precipitate, which is filtered off, washed with water and dried 5 Water and the ethereal layer is Washed With in vacuo at 60' C. to yield 3a-acetoxy-5B-cholanic acid. aqueous 0.1N sodium hydroxide and then with water, dried Step B: Methyl 3a-Acetoxy-SB-Z3-bromocholanate iover magnesium sulfate, filtered and evaporated to yield 23- By substituting 3a-acetoxy-5B-cholanic acid for the 513- nor-24-oxo-5,8-cholane (0.628 g.) in the form of a colorless cholanic acid of example 1, Step A, and following the 40 1 solid. A portion of the compound is purified by chromatogprocedure therein the compound methyl 3a-acetoxy-5B-23- raphy in a mixture of benzene and petroleum over silica gel, 1 bromo-cholanate is obtained. followed by crystallization from ethanol to yield pure 23nor- Step C: 3a-l-iydroxy-trans-A -fifi-cholenic Acid 24-oxo-Sfl-cholane, m.p. l02.5l 04 C. 7 By substituting methyl 360 -acetoxy-5B-23-bromocholanate An ly i for u sa for the methyl 5B-23-bromocholanate of example 1, Step C, 1 C ulat -5 and following the procedure described therein the compound. Found! l 3a-hydroxy-trans-A -5/3-cholenic acid is obtained. Step B: 23-Nor-24-dichloro-5B-cholane Step D: Methyl 3a-Hydroxy-trans-A -5,8-cholenate A solution of 23-nor-24-oxo-5B-cholane (0.627 g., 1.0 3a-HydrQXy-Irans-A -SB-cholenic acid (10 g.) is dissolved mole) in benzene (5 ml.) is added dropwise over a 5-minute in anhydrous methanol (50 ml.) and gaseous hydrogen 50 period to a suspension of phosphorous pentachloride (0.500

chloride (0.5 g.) is bubbled into the mixture. The solution isg., 1.25 mole) in benzene (4 ml.) at 20 C. The mixture is then boiled under reflux for 15 minutes, concentrated in stirred at room temperature for 17 hours and is then poured 5 vacuo while still hot to induce crystallization and then cooled into a mixture of crushed ice and petroleum. The petroleum to room temperature. The product is then filtered, washed layer is washed with aqueous sodium bicarbonate, dried over i with methanol and ri in vac o yield methyl 3a-hydroxymagnesium sulfate, filtered and evaporated at approximately trans-A -5fi-cholenate in the form of a colorless crystalline The crude Product thus Obtained is I ddichloro-SB-cholane (0.710 g.) which could not be crystal- Step E: Methyl 3a-hydroxy-trans-A -5B-cholenate 3-paraliled and WhiCh is Obtained in the form g m toluenesulfonate Step C: ZOa-Ethynyl-SB-pregnane A l i f h l 3 .h d A==.5 A 90 percent sodamide in oil reagent 15 g.) is added to a f (4.00 g.) and para-toluenesulfonyl chloride (2.34 g., 1.20 mol. solution of 'z4'dichioi'or5fi-choiahe 8-) in Xylene ratio) in pyridine 10 ml.) is heated to 60 C. for 24 hours. The 125 ml.) and the mixture heated under reflux for 4 hours with i l ti i then ooled t room temperature d dil t d stirring. The mixture is then cooled to room temperature and slowly with water (30 ml.) while stirring vigorously. A poured into a mixture of crushed ice and petroleum. Some inprecipitate is obtained which is filtered off, thoroughly washed solubles are filtered 0E from the petroleum layer and the or- 5 with water and dried in vacuo to yield methyl 3a-hydroxyganic solution is then washed with water, dried over magnesii. nuns-AJB-cholenate 3-para-toluenesulfona l .um sulfate, filtered and evaporated in vacuo. The residue is Step F: M-Mns-A -SB-ChoIadienic Acid chromatographed in petroleum over a column of basic alu- 5 Methyl 3a-hydroxy-trans-A -5fl-cholenate 3-paramina to yield ZOa-ethynyl-SB-pregnane in the form of a toluenesulfonate is added to a solution of potassium tertiary; crystalline solid (1.60 g.). A portion of the product is Ebutoxide in dimethyl sulfoxide and the mixture heated to a i recrystallized from ethanol to yield pure 20a-ehtynyl-5B- I temperature of l00-l 10 C. for 1 hour. The solution is then pregnane having a melting point of l29l33 C.

f-cooled, diluted with five volumes of cold water and acidified} Analysis for C l-[ with aqueous concentrated hydrochloric acid. The resulting75 Calculated: C, 88.39; H, 11.61;

Found: C, 88.38; H, 11.50. Step D: ZZ-SB-Cholynic Acid 1.68M-Lithium methyl solution (16 ml.) is added to a solui tion of 20a-ethynyl -B-pregnane (1.62 g.) in ether ml.) at

C. causing vigorous evolution of methane and formation of a precipitate. The mixture is exposed to an atmosphere of carbon dioxide at 800 pounds per square inch pressure at room temperature for 17 hours and the mixture is then diluted with ether (100 ml.), washed with aqueous hydrochloric acid and then with water, dried over magnesium sulfate, filtered and evaporated in vacuo. The crude residue thus obtained is crystallized on trituration with petroleum to yield 0.86 g. of 22-5/3-cholynic acid, m.p. l55l60 C. A portion of the product recrystallized from hexane yields pure 22-5B-cholynic acid which also melts at l55l60 C.

Analysis for C l-1 .0,: Calculated: C, 80.85; H, 10.18; Found: C, 81.30; H, 10.39. Step E: Cis-A-5B-Cholenic Acid 22-5B-Cholynic acid (125 mg.) is selectively reduced to the cis-olefin in ethanol solution (10 ml.) employing a prereduced lead-poisoned palladium on calcium carbonate catalyst and hydrogen at atmospheric pressure. The catalyst is filtered off through diatomaceous earth and the filtrate evaporated in vacuo. The crude product is purified by thin-layer chromatography over silica gel using a mixture of chloroform and 6 percent methanol and is then recrystallized from methanol to yield 64 mg. of cis-A -Sfl-chOlenic acid, m.p. 137-l47 C. A portion of the product recrystallized from methanol yields pure cis-A -5fi-cholenic acid having a melting point of i l51-153 C.

Analysis for c,,r-r,,o,: Calculated: C, 80.39; H, 10.68; Found: C, 80.10; H, 10.87. Step F: Cis-A-5B-Taurocholenic Acid Potassium Salt By substituting cis-A -sfi-cholenic acid for the trans-A- SB-cholenic acid of example 1, Step D, and following the procedure described therein the product cisA -SBtaurocholenic acid potassium salt is obtained.

EXAMPLE l4 5B-23-Fluorocholanic Acid Step A: Methyl 5fi-23-Fluorocholanic Acid Potassium fluoride dihydrate (5.23 g.) is suspended in diethylene glycol (80 ml.) and is dehydrated by heating to 240 C. for 10 minutes. The mixture becomes homogeneous and remains so on cooling to 160 C. Methyl 5B-23- bromocholanate (5.00 g.) is then added to the hot reagent, forming a clear solution which is maintained at 160 C. for 20 hours in an atmosphere of nitrogen. The solution is then cooled, diluted with aqueous 0.5N hydrochloric acid (250 ml.) and extracted with two 100 ml. portions of ether. The a combined ethereal extracts are dried over magnesium sulfate, filtered and evaporated in vacuo to yield crude 53-23- fluorocholanic acid. The product thus obtained is then purified by conversion to its corresponding methyl ester according to the method described in the following paragraph and the ester is then converted to the corresponding carboxylic acid according to the method described in Step B, infra. The crude 5B-23-f1uorocholanic acid is converted to its methyl ester by dissolving the acid in methanol (50 ml.) conj taining concentrated sulfuric'acid. The solution is left at room temperature overnight, the methanol is removed in vacuo, the residual oil is dissolved in ether (200 ml.), washed free of sulfuric acid with an excess of aqueous sodium bicarbonate and the ethereal solution dried, filtered and evaporated in vacuo to yield methyl 5B-23-fluorocholanate (4.31 g.). The ester product thus obtained is then subjected to chromatography in benzene (two parts) and petroleum (one part) over silica gel to yield 2.80 g. of a diastereoisomeric mixture of pure methyl 5p-23-fluorocholanate (65 percent), m.p. 89'96' C.

Analysis for C l-F0 Calculated: C, 76.47; H, 10.53; F, 4.83;

16 Found: C, 76.73; H, 10.56; F, 5.20. Step B: 5B-23-Fluorocholanic Acid Methyl 5B-23-fluorocholanate (2.80 g.) is dissolved in hot ethanol (15 ml.) and potassium hydroxide (1.20 g.) in water 15 ml.) is added and the mixture heated to boiling with slow distillation of the ethanol for 20 minutes. The hot solution is acidified with aqueous concentrated hydrochloric acid and the resulting steroid is extracted with two 50 ml. portions of ether. The combined ethereal extracts are then washed with EXAMPLE [5 5,8-23-Chlorocholanic Acid Iodine (0.5 g.) is added to a solution of SB-cholanic acid (25 g.) in sulfuryl chloride ml.) and the mixture is heated under reflux in a water bath at 65 C. for 2 hours and then gradually heated to boiling under reflux for 2 hours. The excess of sulfuryl chloride is removed by distillation in vacuo and the residue is stirred vigorously with water (200 ml.) for 30 minutes. The crude 5fl-23-chlorocholanic acid thus obtained is a colorless solid which is filtered off, washed with hot water, dried in vacuo and recrystallized from ethanol to yield pure 5B-23-chlorocholanic acid.

The products of this invention can be administered in a wide variety of therapeutic dosages in conventional vehicles as, for example, in a sterile pharrnacologically acceptable carrier, by oral administration in the form of a capsule or tablet as well as by intravenous injection. Also, the dosage of the products may be varied over a wide range as, for example, the form of capsules or scored tablets containing 5, 10, 20, 25, 50, 100, 150, 250 and 500 milligrams, i.e., from 5 to about 500 milligrams, of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. These dosages are well below the toxic or lethal dose of the products.

A suitable unit dosage form of the products of this invention can be prepared by mixing 50 mg. of a suitable 5B-taurocholenic acid, SB-taurocholadienic acid or a suitable salt, ester or amide derivative thereof with 144 mg. of lactose and 6 mg. of magnesium stearate and placing the 200 mg. mixture into a No. 3 gelatin capsule. Similarly, by employing more of the active ingredient and less lactose, other dosage forms can be put up in No. 3 gelatin capsules and, should it be necessary to mix more than 200 mg. of ingredients together, larger capsules may be employed. Compressed tablets, pills or other desired unit dosages can be prepared to incorporate the compounds of this invention by conventional methods and, if

desired, can be made up as elixir-s or as injectable solutions by.

methods well known to pharmacists.

lt rs also within the scope of this invention to combine two or more of the compounds of this invention in a unit dosage form or to combine one or more of the compounds with other known hypocholesterolemics and hypolipemics or with other desired therapeutic and/or nutritive agents in dosage unit form.

The following example is included to illustrate the preparation of a representative dosage form:

The trans-A -fi-taurocholenic acid sodium salt is reduced to a No. 60 powder and then lactose and magnesium stearate are passed through a No. 60 bolting cloth onto the powder and the combined ingredients admixed for minutes and then filled into No. 3 dry gelatin capsules.

Similar dry-filled capsules can be prepared by replacing the active ingredient of the above example by any of the other novel compounds of this invention.

It will be apparent from the foregoing description that the SB-taurocholenic acids and Sfl-taurocholadienic acids ofthis invention and their salt, ester and amide derivatives constitute a valuable class of compounds which have not been prepared heretofore. One skilled in the art will also appreciate that the processes disclosed in the above examples are merely illustrative and are capable of wide variation and modification E without departing from the spirit of this invention. What is claimed is:

l. A composition useful in reducing the concentration of cholesterol and lipids in blood serum which comprises as the active ingredient a member selected from the group consisting of:

and

wherein R in both formulas represents hydroxy, lower alkoxy, OM wherein M is a cation derived from a metal of the first group of the Periodic System or NR'R wherein R and R are similar or dissimilar members selected from the group consisting of hydrogen and lower alkyl and, when R is hydroxy, the nontoxic, pharmacologically acceptable salts of the resulting acid; and the dotted line in the three-position of the first formula indicates that that compound may contain an unsaturated double bond within the cholane nucleus and indicates also that the said double bond may be in any other of the several isomeric positions within the nucleus; and a sterile pharmacologically acceptable carrier.

2. A composition useful in reducing the concentration of cholesterol and lipids in blood serum which consists of from 5 to 500 mg. of rrans-A -5B-taurocholenic acid, sodium salt in combination with a sterile pharmacologically acceptable car-v ner.

I t I i 

2. A composition useful in reducing the concentration of cholesterol and lipids in blood serum which consists of from 5 to 500 mg. of trans- Delta 22-5 Beta -taurocholenic acid, sodium salt in combination with a sterile pharmacologically acceptable carrier. 